Production of cell suspensions

ABSTRACT

Methods are provided for producing cell suspensions suitable for engraftment and cell products suitable for infusion into a human patient. Some methods include (a) culturing a first population of cells that contains multiple cell types, including a first sub-population of cells of a type suitable for engraftment, under conditions that cause the expansion of the first sub-population of cells, wherein the population of cells further comprises a second sub-population of cells that either inhibit the expansion, or gwo less well under the culture conditions than the cells of the first sub-population, and that are capable of enhancing engraftment; (b) before, during, or following the culturing of step (a), removing from the population cells of the second sub-population; (c) preserving cells of the second sub-population; and (d) following expansion of the cells of the first sub-population, combining cells fo the first sub-population with cells of the preserved second sub-population.

BACKGROUND OF THE INVENTION

This invention relates to the production, by expansion and selectionmethods, of cell populations that can be infused into patients, forexample, to engraft patients, e.g., cancer patients who have hadtreatment that resulted in the depletion of their bone marrow. Otheruses of such cell populations include supplying cells as supplements forgenetic defects, as elements for regeneration of tissue, as carriers oftherapeutic genes, and as agents used for immune enhancement.

In the case of engraftment, cancer patients can have their bone marrowreconstituted by the administration of bone marrow obtained from donors,or by the administration of suspensions of relatively immature, andtherefore, pluripotent, cells, including stem cells, obtained from othersources, such as umbilical cord blood. Methods have been devised toexpand such cells in culture, while selecting the desired, or “target”cells, such as stem cells. A method for achieving this is described inKraus, U.S. Pat. No. 5,925,567, hereby incorporated by reference. Suchsystems typically employ cytokines and other growth factors to ensurethat the resulting cells suspension is enriched in the target cell type.In addition, such methods have employed supporting cells such as stromalcells, which can enhance the expansion of the target cells.

SUMMARY OF THE INVENTION

The invention features a method for producing an engraftable cellsuspension, wherein the method includes the steps of:

a) culturing a first population of cells that contains multiple celltypes, including a first sub-population of cells of a type suitable forengraftment, under conditions that cause that expansion of the firstsub-population of cells, wherein the population of cells furthercomprises a second sub-population of cells that either inhibit theexpansion, or grow less well under these conditions than the cells ofthe first sub-population, and that are capable of enhancing engraftment;

b) before, during, or following the culturing of step (a), removingcells of the second sub-population;

c) preserving the removed cells of the second sub-population; and

d) following expansion of cells of the first sub-population, combiningcells of the first sub-population with cells of the preserved secondsub-population to form the engraftable cell suspension.

In certain embodiments, step (d) is performed after expansion of thefirst sub-population of cells has been completed. The method can furtherinclude the removal of dead cells, and can also include the step ofintroducing a third sub-population of engraftment-enhancing cells orexpansion-enhancing cells to the suspension, at any stage in theprocess. In cases where the cells of the third sub-population enhanceexpansion of the first sub-population, the cells of the thirdsub-population are added to the culture containing the firstsub-population of cells prior to the completion of the expansion, inorder to enhance that process. The cells of the second sub-populationthat are removed initially can be expanded prior to being re-combinedwith the cells of the first sub-population. Removal of the cells of thesecond sub-population can be effected by use of selection elements, e.g.antibodies, cell adhesion molecules (CAMs), and/or ligands to variousgrowth factors. Positive or negative selection can be used, e.g. asdescribed in U.S. Pat. No. 5,925,567. Repeated or continuous selectionof one or more sub-populations can be performed, with the criteria forselection being varied over time if desired.

The invention takes advantage of the discovery that some sub-populationsof cells, e.g., cells that express the surface antigen CD14, whileundesirably inhibiting the expansion of desired cells, e.g. CD45+ cellssuch that their removal enhances expansion, may enhance characteristicsof the engraftable cell suspension if added back following expansion. Afurther important insight is that sub-populations that are removed,temporarily, can be expanded prior to being added back to the cellsuspension, under conditions which may be different from those in theoriginal culture, and more conducive to expansion of the removedsub-population. Because a starting cell culture may contain a number ofdifferent cell types, any of which can be removed at any stage of theprocess, expanded, and added back in any proportion, the characteristicsof the final cell suspension can be adjusted infinitely using themethods of the invention.

Certain cell sub-populations enhance the growth of the primary cultureand/or the sub-population, and/or enhance the properties (potency,efficacy, etc.) of the cell suspension. Growth enhancement and/orenhancement of the properties of the final cell suspension can beobtained by recombining the primary culture, sub-populations and/orprogeny of sub-populations at various times during or after expansion.

Other features and advantages of the invention will be apparent from thedetailed description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the effect of various antibody cocktails onthe expansion potential of a cell culture.

FIG. 1A is a graph illustrating the effect of various concentrations ofCD14+ cells on the expansion potential of a cell culture.

FIG. 2 is a highly enlarged diagrammatic view of a system for positiveselection of a target cell. FIG. 2A is a highly enlarged diagrammaticview of a system for negative selection of a non-target cell.

FIGS. 3 a-3 d are schematic diagrams of various modes of operation ofsystems according to different embodiments of the invention.

DETAILED DESCRIPTION

The invention features methods of forming cell products having definedprofiles by selecting a sub-population from a primary culture, or froman initial cell suspension not in culture, and using the sub-population,a component of the sub-population, and/or the primary culture to makeone or more cell products. In some cases, the sub-population mayultimately be added to the primary culture and/or the cell suspension toform the final product. Examples of initial cell suspensions includebone marrow, cord blood, peripheral blood, and suspensions of cellsobtained from human mesodermal, ectodermal, and endodermal tissues, suchas pancreatic, hepatic, neural, nephrotic, dermal, muscle and cardiactissue. Examples of primary cultures include cultures of cells of a typesuitable for engraftment, such as hematopoietic stem cells.

The methods of the invention may include forming cell products bycombining cells of the sub-population with other cells. For example, thesub-population may be returned to the primary culture after furtherexpansion of the primary culture. Alternatively, the sub-population maybe cultured separately and eventually returned to the primary culture,or its progeny may be returned to the primary culture (either with orwithout intervening further expansion of the primary culture). A secondsub-population may be removed from the first sub-population, and thenthe first or second sub-population may be returned to the primaryculture, either before or after further expansion of the primary cultureand/or the sub-population that is being returned. The sub-population canalso be added to a cell suspension from which the primary culture or thesub-population was obtained, e.g., one of the initial cell suspensionsdiscussed above.

In other implementations, methods include, during expansion of asub-population, adding cells of the same type as those in thesub-population to the sub-population to fuel expansion of thesub-population, and/or removing a sub-population and combining it withother cells not of the primary culture.

In some implementations, in addition to the selection steps discussedabove, an undesirable sub-population is removed from the primary cultureand discarded.

Cell products may be produced by providing a primary culture or initialcell suspension, and performing one or more selection steps (e.g.,selection after cell division or proliferation has begun) using one ormore antibody cocktails that remove a predetermined sub-population fromthe primary culture. The effect of various antibody cocktails on theexpansion potential of a particular primary culture is shown in FIG. 1.In the experiment illustrated in FIG. 1, selection was performed priorto cell expansion. However, as will be discussed below in detail,selection with the antibody cocktails can be performed at any time andcan be performed repeatedly before, during and after cell division.

Cell products may also be produced by (a) culturing a first populationof cells that contains multiple cell types, including a firstsub-population of cells, under conditions that cause the expansion ofthe first sub-population of cells, and (b) during or followingexpansion, removing from the first population a second sub-population ofcells that are generated by the first sub-population, the infusable cellsuspension including cells of the second sub-population. In some cases,the infusable cell suspension will be substantially free of cells of thefirst population, e.g., if such cells are not suitable for infusion. Insome cases, for example in the case in which the first sub-populationincludes embryonic stem cells, the removing step is performed duringexpansion, and may be performed substantially continuously.

The composition of the antibody cocktail may be changed as time passesduring cell expansion. For example, surface molecules expressed atdifferent time points on different cells can be identified, andselection in or out can be based on this. Reselection with differentantibody cocktails can be performed substantially continuously, or on anintermittent basis.

The proportions of different cells in the cell product can be adjusted,e.g., by adjusting antibody concentrations so that only a givenproportion of non-target cells are selected out, and/or by changing thenumber and/or identity of the antibody or antibodies in the cocktail.

The antibody or antibodies in the cocktail can be selected to increasethe fidelity of the product since some cell surface markers are sharedby different cell types. For instance, CD45 positive cells bear at leastseveral CD45 epitopes that represent unique subpopulations ofleukocytes. As a result, a CD45 pan leukocyte marker can be used toselect in or out all leukocytes, whereas CD45RA and CD45RO can be usedto select in or out specific leukocyte subpopulations.

Proportions of cell types can also be varied by recombining the primaryculture with sub-populations and/or progeny of sub-populations, duringor after expansion.

Repeated or continuous selection of one or more sub-populations from theprimary culture may be performed, the criteria for the selection beingvaried over time. For example, a particular sub-population of cells maybe removed from the primary culture during initial selection steps, toenhance expansion of the primary culture, but not removed from theprimary culture during later selection steps, to enhance the propertiesof the final cell product.

Producing Cell Products

Some examples of suitable techniques for producing cell products thatare suitable for infusion into a patient are discussed in detail below.

During cell culture, a first selection step may include selecting out afirst sub-population that expresses CD14. This may be advantageous, asCD14 appears to inhibit the expansion of some cell populations, e.g.,CD45+ cells, CD34+ cells and CD34+/38− cells, as shown in FIG. 1A.

From the first sub-population, a narrower, second sub-population thatco-expresses CD14 and CD38 maybe selected. This second sub-population isreturned to the primary culture, after further expansion has occurred,or is added to the finished cell product. This may be desirable forseveral reasons.

First, the first sub-population may be undesirable in the culture, whilethe second sub-population is desirable. For example, the firstsub-population may have a negative impact on culture kinetics, while thesecond sub-population may have an advantageous impact on culturekinetics and/or may favorably affect the potency of the suspension.Since the two sub-populations share a common selection marker, in orderto separate the second sub-population from the first sub-population, sothat the second sub-population may be returned to the culture, it isnecessary to perform the two sequential selection steps described above.In this manner, the second sub-population can be isolated and returnedto the culture without returning the undesirable first sub-population.If desired, e.g., if the first sub-population is detrimental to culturekinetics but advantageous to potency, the first sub-population can bereturned at the conclusion of the expansion process.

If desired, the first and/or second sub-populations may be culturedseparately prior to returning the second sub-population to the primaryculture or adding it to the final cell product.

A sub-population can be removed from the primary culture and thenreturned after further expansion of the primary culture. This is donewhere the sub-population has an undesirable effect on expansion of theprimary culture, but is useful in the final product. For example, CD14+cells tend to inhibit expansion of a population of CD34+/CD38− cells,but may provide short-term support during the engraftment phase afterdelivery of the final product to a patient. The sub-population may bereturned to the primary culture without further expansion of thesub-population, if desired.

The sub-population can also be cultured in a separate culture and thenlater returned to the primary culture. For example, again usingCD34+/CD38− as the primary culture, a sub-population of CD7lymphopoietic precursor cells can be removed and cultured under cultureconditions that favor lymphopoietic cells. The expanded CD7sub-population is then returned to the primary culture, e.g., to helpreconstitution of lymphopoiesis of the immune system of the patient andthereby increase the kinetics of immune reconstitution.

If desired, in the above example CD3 cells can be separated out of theexpanded CD7 sub-population before it is returned to the primaryculture, to remove more differentiated lymphopoietic T-cells that maycause graft versus host disease (GVHD) in a patient.

During methods involving expansion of a sub-population, cells of thesame type as those in the sub-population can be added to thesub-population culture to fuel expansion of the sub-population. Thesecells can be obtained by repeated selection from the primary culture asthe primary culture continues to expand.

In some cases, rather than returning the sub-population to the primaryculture, the sub-population can be added to a starting material fromwhich the primary culture was obtained. For instance, the primaryculture such as CD34+/CD38− can be obtained from cord blood andexpanded, and subsequently added back to the cord blood to form afinished product. This would allow cord blood to be delivered to a largepatient in need of a large number of CD34+/CD38− cells as well as otherinherent constituents of cord blood.

It may also be desirable to remove cells of a sub-population from aprimary culture, and add types other cells not of the primary culture toform a final product. For example, CD34+/CD38− cells and dendritic cellsmay be isolated from cord blood. The dendritic cells, which aredifficult to expand, would be stored, while the CD34+/CD38− cells arecultured in a primary culture under conditions that will producedendritic cells. The dendritic cells are then separated from the primaryculture as a sub-population, and added to the dendritic cells that wereisolated originally, to form a final product consisting of dendriticcells.

Dead cells may be removed from the primary culture and/or asub-population separated from the primary culture and discarded. Theresulting purified primary culture and/or sub-population can then beused as separate cell products or one combined cell product. Forexample, dead cells can be removed from a primary culture of CD34+/CD38−cells or a sub-population derived from this primary culture, and thepurified culture or sub-population can be used as a final product.

Removal of dead cells from the primary culture has several advantages.Dead cells could impact the safety of the product, e.g., by causingundesirable reactions by the patient's immune system, or the potency ofthe other cells, and components released by dead cells could inhibitexpansion or cause differentiation or apoptosis of the target cells.This is important because the resulting product will have a higher valuebecause of higher purity and potency (both clinical and perceivedimprovement in potency and safety). In some preferred cell products, atleast 85% of the cells present in the product are viable cells, morepreferably at least 95% of the cells are viable.

Selection Techniques

Preferred methods include selecting a sub-population of cells from cellsin the primary culture, concurrently with proliferation, intermittentlyduring proliferation or following proliferation. Cell proliferation andcell selection can be carried out using an almost infinite variety ofdifferent techniques and settings, of which only a few are describedbelow by way of example. Many other techniques will be readily perceivedby those skilled in the art, for example selection by flow cytometry,and selection by using chemical agents to kill unwanted cells. Selectionmay be performed using selection elements against cell surface markers.Positive or negative selection may be used, e.g., as described in U.S.Pat. No. 5,925,567, the disclosure of which is incorporated herein byreference.

The preferred selection methods used in the invention can broadly beclassed as positive selection (providing a selection element having anaffinity for target cells) and negative selection (providing a selectionelement having an affinity for non-target cells). These two selectiontechniques, used alone or in combination, allow cells to be removed fromthe primary culture whenever desired, and also allow cells to bereselected from subpopulations to produce additional, narrowersubpopulations.

An example of a positive selection technique is illustrateddiagrammatically in FIG. 2. Briefly, one or more anti-dextran conjugatedantibodies specific for the predetermined target population isintroduced into the culture. After a specified incubation time, amagnetic dextran iron particle colloid is introduced into the cellsuspension. A Cell/Antigen/Antibody/Anti-dextran/Dextran/Iron Complexforms. This complex is then passed through a magnetic field. Positivelyselected cells remain in the magnetic field while cells which do nothave the iron conjugated complex are removed. After capture and rinsingthe magnetic field is removed and the positively selected predeterminedtarget population is returned to the nutrient medium.

An example of a negative selection technique is illustrateddiagrammatically in FIG. 2A. Briefly, one or more anti-dextranconjugated antibodies specific for a predetermined population which isnot of the predetermined target population is introduced into theculture. After a specified incubation time, a magnetic dextran ironparticle colloid is introduced into the cell suspension. ACell/Antigen/Antibody/Anti-dextran/Dextran on Complex forms. Thiscomplex is then passed through a magnetic field, removing cells not ofthe predetermined target population from the nutrient medium. Thepredetermined target population is collected downstream and returned tothe nutrient medium.

Clearly, many other techniques can be utilized for both positive andnegative selection, as long as the desired affinity is provided by theselection element.

The selection element can include other components in addition to theantibody molecules that are used to perform the selection (the“selection molecules”), e.g., a solid support to which the selectionmolecule is bound. The solid support can be formed of a material thatwill aid in performing the selection or in maintaining the selectionmolecules in a desired position or introducing and removing them fromthe system. For example, as described above with reference to FIG. 2,the selection molecule can be bound to iron or other magnetic particlesto allow the selected cells to be easily removed from the system byapplication of a magnetic field and then collected by removal of themagnetic field. Alternatively, the selection molecules can be bound ontothe wall of a vessel containing the nutrient medium, or of a chamberthrough which the nutrient medium flows during the method. Glass orother inert, impermeable beads can also be used as a solid support. Ifbeads or other particles are used, they can be provided in a packedconfiguration, through which the nutrient medium flows, or can beintroduced into the system in a loose form, suspension, or in anydesired type of array. As will be readily understood, a wide variety ofother solid supports can be used.

As shown in FIGS. 3-3D, the selection element can be used in a varietyof modes of operation in which nutrient media is supplied to and removedfrom the system in different manners. These modes of operation rangefrom a selective batch culture (FIG. 3), in which nutrient media issupplied at the beginning of cell proliferation and is neither added tonor removed, to continuous flow or recycled flow cultures (FIGS. 3C and3D, respectively) in which either fresh or recycled nutrient media flowsthrough the system substantially continuously. These alternative modeswill be discussed in detail below.

In a selective batch culture (FIG. 3), a nutrient medium is introducedinto a vessel, and a starting sample of cells is also introduced intothe vessel. During cell proliferation, nutrient medium may or may not beexchanged. However, selected cells are physically selected, i.e.,separated from other cells in the nutrient medium by binding to aselection element, either continuously, intermittently or following cellproliferation These selected cells may be cells of a target population,if positive selection is used, or unwanted cells, if negative selectionis used. Dual (positive and negative) selection can be accomplished byproviding positive selection molecules on the surface of the vessel,beads, baffles, impellers, etc. while removing unwanted cells bynegative selection. Alternatively, cells may be positively or negativelyselected outside of the culture vessel and then returned.

The selective semi-batch (3A) and selective fed batch (3B) modes ofoperation are similar to the selective batch mode with regard topositive and negative selection The significant difference between thesethree modes is in the treatment of the nutrient medium. While in thebatch mode the volume of the medium remains constant and the medium isnot refreshed (it may be supplemented), the semi-batch mode allows for apartial refreshment of spent medium with new medium and the fed batchmode allows for an incremental increase in the medium volume over time.

Cell growth and selection can also be performed in a continuous (FIG.3C) or recycling (FIG. 3D) mode of operation. In these two modes, thesystem includes a chamber having an inlet and an outlet, and nutrientmedia is caused to flow through the chamber from the inlet to theoutlet. In continuous mode, new nutrient media flows through the chamberfrom a source or reservoir, while in recycling mode the same nutrientmedia is cycled through the chamber repeatedly. If desired, a system canbe configured to be run alternatively in either continuous or recyclingmode. Any desired selection element can be used in these modes ofoperation.

EXAMPLE 1

Using a 9 antibody (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, andGlyA) negative selection cocktail (Stemcell Tech) as a normalizedcontrol we tested the relationship between the initial antibodycombination and the Total Cell and CD34+/CD38− cell output at day 7 postinoculation of lineage depleted Umbilical Cord Blood. Cultures containedFlt-3, SCF, and Tpo at 100 ng/ml each.

Referring to FIG. 1, the selection cocktails represented deviated fromthe 9 antibody control cocktail (3^(rd) set of bars) in several ways.From left to right, cocktails 1-14 were no GLYA or CD24 with the other 7antibodies (Abs) of the control cocktail plus CD38 (N=2), no CD19 withthe other 8 Abs (N=4), all 9 Abs (control; N=10), no CD16 with the other8 Abs (N=4), no CD56 with the other 8 Abs (N=4), no CD66b with the other8 Abs (N=4), no CD14 with the other 8 Abs (N=4), no CD2 with the other 8Abs (N=4), no GlyA with the other 8 Abs (N=4), no CD24 with the other 8Abs (N=9), no CD3 with the other 8 Abs (N=9), no GlyA or CD24 (N=4) withthe other 7 Abs, no GlyA or CD3 with the other 7 Abs (N=4), and no GlyAor CD3 or CD24 with the other 6 Abs (N=8), respectively.

This combinatorial approach reveals the differential generation, in boththe quantity and identity, of the process output. The selectiveexpansion of distinct populations using various antibody cocktailselection strategies is critical given the impact of distinctpopulations on the production of target populations such as CD34+/CD38−cells. One example of this potential negative impact on the relativeproduction of CD34+/CD38− cells can be ascribed to the presence of CD14+cells in the configuration, no CD14 with the other 8 Abs (cocktail #7).This cocktail amplified CD34+/CD38− at 81.6% of the 9 antibody control(cocktail #3). Another example is the independent observation that thelack of CD38+ cells, in the presence of both GlyA+ and CD24+ cells, inthe CD38 depleted culture (cocktail #1) resulted in an amplification inCD34+/CD38− cells that was 66.9% of the 9 antibody control. This lastobservation is in contrast to observations regarding cocktails #9, #10,#12, and #14 that each lacked GlyA or CD24 or both. In these instancesthe amplification of CD34+/CD38− was increased by 113.1, 124.9, 107.2,and 126.4 respectively, relative to the 9 antibody control

EXAMPLE 2

This study was initiated based on the empirical finding that CD34+/CD38−cells expanded with less amplitude, 84.6% N=4) of the control (N=10), ifthe Lin-cocktail (CD2/CD3/CD14/CD16/CD19/CD24/CD56/CD66b/GlyA-control)used to generate the inocula did not contain the antibody CD14. Withthis result we proceeded to test, in a dose dependent manner, the effectof CD14+ cells on the expansion of highly selected CD34+/CD38−populations. Umbilical Cord Blood was obtained and separated by magneticmeans whereby CD14+ cells were first isolated by positive magneticselection (Dynal) the cell suspension was then treated to isolate thetarget population using the 9 antibody control cocktail described above(StemCell Tech), except for the exclusion of the CD14 antibody.Approximately equal fractions containing a combination of CD45+(5.00×10⁵), CD34+ (6.84×10⁴), CD34+/CD38−/Lin- (2.19×10⁴) cells from thesame specimen were seeded with increasing numbers of CD14+ cells. TheLin- to CD14+ ratios were 1:0, 50:1, 1:1, and 1:3, respectively. Withthe four supplemental CD14+ cell values the initial number of CD45+cells in each culture was 5.00×10⁵, 5.10×10⁵, 1.00×10⁶, and 2.00×10⁶,respectively. On day 7 of the cultures, cells were sampled to determinethe absolute number of cells in each of three phenotype compartments,namely CD45, CD34 and CD34+/CD38− cells, relative to their initialnumbers.

The results of this testing are shown in FIG. 1A. Each of the threecompartments was sensitive to the presence of supplemental CD14+ cellsat as low as a one (1) CD14+ cell per initial fifty (50) Lin- cells (or,0.5% supplemented). Furthermore, increased concentrations of CD14+ cellsled to the near abrogation of growth in the CD34+ and CD34+/CD38−compartments, with the mature CD45+ compartment being more or lessannihilated at the highest CD14+ cell ratio.

This result demonstrates that specific populations, such as the CD14+cells, can significantly impact the ability to effectively expand thetarget population, in this case CD45+ cells. Despite this inhibitoryeffect, CD14+ cells may improve the effect of the graft on a patient,and thus it may be desirable to recombine the CD14+ cells with thetarget cells after expansion of the target population is complete.

Other embodiments are within the scope of the following claims.

1. A method for producing an engraftable cell suspension, said methodcomprising the steps of: a) culturing a first population of cells thatcontains multiple cell types, including a first sub-population of cellsof a type suitable for engraftment, under conditions that cause theexpansion of said first sub-population of cells, wherein said firstpopulation of cells further comprises a second sub-population of cellsthat either inhibit said expansion, or grow less well under saidconditions than the cells of said first sub-population, and that arecapable of enhancing engraftment; b) before, during, or following theculturing of step (a), removing from said first population cells of saidsecond sub-population; c) preserving cells of said secondsub-population; and d) following expansion of said cells of said firstsub-population, combining cells of said first sub-population with cellsof the preserved second sub-population to form said engraftable cellsuspension.
 2. The method of claim 1, wherein step (d) is performedafter expansion of said first sub-population is completed.
 3. The methodof claim 1, wherein the method further includes removal of dead cells.4. The method of claim 1, wherein step (d) further comprises introducinga third sub-population of engraftment-enhancing cells orexpansion-enhancing cells or potency-enhancing to said engraftable cellsuspension.
 5. The method of claim 4, wherein said third sub-populationof cells are added prior to completion of the expansion of said firstsub-population of cells, and wherein said third population of cellsenhance the expansion of the first sub-population of cells.
 6. Themethod of claim 4, wherein said third sub-population of cells enhancesthe potency of the engraftable cell suspension.
 7. The method of claim1, further comprising the step of expanding the cells of the secondsub-population prior to step (d).
 8. The method of claim 1, whereincells of the second sub-population are removed in step (b) usingselection elements that recognize surface markers on said cells.
 9. Amethod of producing a cell suspension that is suitable for infusion intoa human patient comprising: (a) culturing a first population of cellsthat contains multiple cell types, including a first sub-population ofcells, under conditions that cause the expansion of said firstsub-population of cells; (b) before, during or following expansion,removing from said first population a second sib-population of cellsthat have an inhibitory effect on expansion of the first sub-populationof cells or grow less well under said conditions than the cells of saidfirst sub-population; and (c) after further expansion of the firstpopulation of cells, combining cells of said second sub-population withcells obtained by expansion of said first sub-population.
 10. The methodof claim 9, wherein step (c) is performed after expansion of said firstsub-population is completed.
 11. The method of claim 9, furthercomprising removing dead cells.
 12. The method of claim 9, wherein step(c) further comprises introducing a third sub-population of cells tosaid cell suspension.
 13. The method of claim 12, wherein said thirdsub-population of cells are added prior to completion of the expansionof said first sub-population of cells, and wherein said third populationof cells enhance the expansion of the first sub-population of cells. 14.The method of claim 12, wherein said third sub-population of cellsenhances the potency of the cell suspension.
 15. The method of claim 9,further comprising the step of expanding the cells of the secondsub-population prior to step (c).
 16. The method of claim 9, whereincells of the second sub-population are removed using selection elementsthat recognize surface markers on said cells.
 17. A method of producinga cell suspension product that is suitable for infusion into a humanpatient comprising: (a) acquiring an initial cell suspension containingmultiple cell populations, (b) selecting a population of target cellsfrom said initial cell suspension, (c) culturing said target cells underconditions that cause the expansion of said target cells, (d) combiningat least a portion of the product of step (c) with the remainder of saidinitial cell suspension to form the cell suspension product.
 18. Themethod of claim 17 wherein said initial cell suspension is selected fromthe group consisting of bone marrow, cord blood, and peripheral blood.19. The method of claim 17 wherein said initial cell suspension containscells obtained from human mesodermal, ectodermal, and endodermaltissues.
 20. The method of claim 19 wherein cells of said initial cellsuspension are obtained from tissue selected from the group consistingof pancreatic, hepatic, neural, nephrotic, dermal, muscle and cardiac.21. The method of claim 17 wherein said initial cell suspension containsembryonic stem cells.
 22. The method of claim 21 wherein said targetcells are selected from the group consisting of CD34+/CD38− multipotentprogenitors, CD34+/CD7+ common lymphoid progenitors, CD34+/CD33+ commonmyeloid progenitors, and combinations thereof.
 23. The method of claim17 further comprising infusing the product of step (d) into a humanpatient.
 24. The method of claim 17 further comprising, during step (c),removing from the culture a sub-population of cells that have aninhibitory effect on expansion of the target cells or grow less wellunder the culture conditions than said target cells.
 25. The method ofclaim 24 further comprising, after further expansion of the target cellculture, combining cells of said sub-population with the cultured targetcells or the initial cell suspension.
 26. The method of claim 25 furthercomprising expanding said sub-population.
 27. The method of claim 17further comprising selecting, from either said initial cell suspensionor said population of target cells, a sub-population of cells other thansaid target cells.
 28. The method of claim 27 further comprising, duringstep (d), combining at least a portion of the cells of thesub-population with the initial cell suspension.
 29. A method ofproducing a cell suspension that is suitable for infusion into a humanpatient comprising: culturing a first population of cells that containsmultiple cell types, including a first sub-population of cells, underconditions that cause the expansion of said first sub-population ofcells; and during or following expansion, removing from said firstpopulation a second sub-population of cells that are generated by saidfirst sub-population; wherein the cell suspension comprises said secondsub-population.
 30. The method of claim 29 wherein the removing step isperformed during expansion.
 31. The method of claim 30 wherein removalis performed substantially continuously.
 32. The method of claim whereinsaid cell suspension is substantially free of cells of said firstpopulation.